1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.

Abstract

BACKGROUND:

In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery.

RESULTS:

AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis.

CONCLUSION:

AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.

AAV2 induced DNA laddering/degradation in multiple breast cancer cell lines. (A) MCF-7, (B) MDA-MB-468, (C) MDA-MB-231 and (D) nHMECs monolayer cultures were synchronized in G1, followed by infection with AAV2 at an MOI of 0.02. Cell pellets were collected each day over a seven day period for the breast cancer cell lines, and over a five day period for the nHMECs. The breast cancer cell lines were passaged 1:2 on day 2 and day 5, and on day 2 for nHMECs. DNA laddering assays were performed by isolating low-molecular weight DNA using protocols described herein. Twenty micrograms of DNA were resolved in a 1% agarose Tris-Borate-EDTA gel and stained with ethidium bromide. Results shown are representative of three individual experiments. t, time; +, AAV2 infected; -, control. DNA ladders are indicate the following base-pair fragments starting at the top: 23941, 9416, 6557,4361, 2322, 2027, 1353, 1073, 872, 603, 310. (E) nHMECs cultured from multiple patient breast tissue biopsies were used to prepare whole cell extracts. Five micrograms of the extract was used for western blot analysis. Cytokeratin markers Involucrin, K10 and K14 were detected using antibodies as described in Materials and Methods.

Cell viability as a measure of AAV2 induced cell death in the breast cancer cell lines. (A) MCF-7, (B) MDA-MB-468 and (C) MDA-MB-231 cells were infected with AAV2. Trypan blue exclusion was used to determine the growth inhibitory effect of AAV2 on the monolayer cultures of the different breast cancer cell line cultures. Results shown are representative of three individual experiments. t = time. Arrows indicate days when the cells were passaged at a ratio of 1:2.

AAV2 induction of apoptosis/cell death in the different breast cancer lines correlates with Rep protein expression. For detecting Rep proteins in Western blots, total protein extracts were prepared as we have previously described [13,48]. Sixty micrograms of total protein extracts from AAV2 infected and control (A) MCF-7, (B) MDA-MB-468, (C) MDA-MB-231 and (D) nHMECs monolayer cultures were resolved in a 7.5% SDS-polyacrylamide gel electrophoresis gel and detected with an AAV2 Rep-specific antibody (Progen). Results shown are representative of three individual experiments. t, time; +, AAV2 infected; -, control. Double asterisks (**) indicate dimer form of protein, single asterisk (*) indicates the monomer form.

Determining the ability of nHMECs to undergo chemically induced and Rep78 induced apoptosis. (A) nHMECs were treated with either staurosporine (1 μM) to activate the instrinsic pathway, and a combination of cycloheximide (CHX) (10 μg/ml) and Tumor Necrosis Factor-Alpha (TNFα) (20 ng/ml) to activate the extrinsic pathway of apoptosis. Cells were treated for 24 h, harvested prepared for FACS analysis, and 106 cells were analyzed by flow cytometry. Top panel denotes histograms of the DNA distribution representing percentages of cells in the G1, S and G2 phases of the cell cycle denoted in the bottom panel, as determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. (B) Staurosporine treatment induced cleavage of caspase-9 and CHX/TNFα treatment induced cleavage of caspase-8 as determined from western blot analysis. (C) Calcium-phosphate transfection of the cloned Rep78 expression construct under CMV promoter control into nHMECs. Untransfected control, GFP-only controls (+GFP), and GFP vector and CMV-Rep78 construct co-transfected cells (+GFP/Rep78). Top panel denotes histograms of the DNA distribution representing percentages of cells in the G1, S and G2/M phases of the cell cycle denoted in the bottom panel, as determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. Analysis of transfected cells analyzed at 48 h and 7 days post-transfection.

AAV2 induction of apoptosis in MCF-7 cells results in PARP cleavage independent of caspase activation. MCF-7 monolayer cell cultures were synchronized in G1, followed by infection with AAV2. Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of caspases and their cleavage/activation was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected MCF-7 and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect the 35 kDa pro-caspase form of caspase-6, proteins were resolved in a 10% SDS-PAGE gel and detected with a rabbit polyclonal antibody (Cell Signaling Technology). To detect both the pro- and cleaved forms of caspase-7, caspase-8 and caspase-9, proteins were resolved in a 10% SDS-PAGE gel. The 35 kDa pro-caspase form of caspase-7 was detected with a mouse monoclonal antibody (Cell Signaling). The 58 and 56 kDa forms of caspase-8 were detected with a mouse monoclonal antibody (Alexis Biochemicals). The 47 kDa pro-caspase form of caspase-9 was detected with a rabbit polyclonal antibody (Cell Signaling). To detect the pro- (116 kDa) and cleaved- (89 kDa) forms of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).

AAV2 induction of apoptosis in MDA-MB-468 cells results in PARP cleavage following activation of caspases of the intrinsic pathway. MDA-MB-468 monolayer cell cultures were synchronized in G1, followed by infection with AAV2. Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of caspases and their cleavage/activation was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control MDA-MB-468 cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect the 35 kDa pro-caspase form of caspase-3, proteins were resolved in a 10% SDS-PAGE gel and detected with caspase-3 rabbit monoclonal antibody (Cell Signaling Technology). To detect the17-kDa cleaved caspase-3 forms, proteins were resolved in a 15% SDS-PAGE gel and detected with rabbit polyclonal antibody against cleaved caspase-3 (Cell Signaling Technology). To detect the 35 kDa pro-caspase form of caspase-6, proteins were resolved in a 10% SDS-PAGE gel and to detect the 15 kDa cleaved form of caspase-6, proteins were resolved in a 15% SDS-PAGE gel and detected with a rabbit polyclonal antibody (Cell Signaling Technology). To detect both the pro- and cleaved forms of caspase-7, caspase-8 and caspase-9, proteins were resolved in a 10% SDS-PAGE gel. The 35 kDa pro-caspase form and the 30 kDa and 20 kDa cleaved forms of caspase-7 was detected with a mouse monoclonal antibody (Cell Signaling). Caspase-8 was detected with a mouse monoclonal antibody (Alexis Biochemicals). The 47 kDa pro-caspase and 35 kDa cleaved of caspase-9 was detected with a rabbit polyclonal antibody (Cell Signaling). To detect the pro- (116 kDa) and cleaved- (89 kDa) forms of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).

AAV2 induction of apoptosis/cell death in MDA-MB-231 cells results in activation of caspases of both the intrinsic and extrinsic pathways but not PARP cleavage. Left panel: MDA-MB-231 monolayer cell cultures were synchronized in G1, followed by infection with AAV2. Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of caspases and their cleavage/activation was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control MDA-MB-468 cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect the 35 kDa pro-caspase form of caspase-3, proteins were resolved in a 10% SDS-PAGE gel and detected with caspase-3 rabbit monoclonal antibody (Cell Signaling Technology). To detect the17-kDa cleaved caspase-3 forms, proteins were resolved in a 15% SDS-PAGE gel and detected with rabbit polyclonal antibody against cleaved caspase-3 (Cell Signaling Technology). To detect the 35 kDa pro-caspase form of caspase-6, proteins were resolved in a 10% SDS-PAGE gel and to detect the 15 kDa cleaved form of caspase-6, proteins were resolved in a 15% SDS-PAGE gel and detected with a rabbit polyclonal antibody (Cell Signaling Technology). To detect both the pro- and cleaved forms of caspase-7, caspase-8 and caspase-9, proteins were resolved in a 10% SDS-PAGE gel. The 35 kDa pro-caspase form and the 30 kDa/20 kDa cleaved forms of caspase-7 was detected with a mouse monoclonal antibody (Cell Signaling). The pro-caspase and cleaved 28 kDa form of caspase-8 was detected with a mouse monoclonal antibody (Alexis Biochemicals). The 47 kDa pro-caspase and 37 kDa/35 kDa cleaved forms of caspase-9 were detected with a rabbit polyclonal antibody (Cell Signaling). To detect the pro- (116 kDa) form of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).

AAV2 infection of nHMECs does not result in activation of caspases of either the intrinsic and extrinsic pathways of apoptosis. nHMEC monolayer cultures were synchronized in G1 as described, followed by infection with AAV2. Cell pellets were collected each day over a 5 day period. Cells were passaged 1:2 on day 2. Detection of caspases and their cleavage/activation was performed by Western blotting. Sixty micrograms of total protein extracts from AAV2 infected and control nHMEC cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. To detect caspase-8 proteins were resolved in a 10% SDS-PAGE gel and detected with a mouse monoclonal antibody (Alexis Biochemicals). To detect the 35 kDa pro-caspase form of caspase-3, proteins were resolved in a 10% SDS-PAGE gel and detected with caspase-3 rabbit monoclonal antibody (Cell Signaling Technology). To detect the pro- (116 kDa) form of PARP, proteins were resolved in a 7.5% SDS-PAGE gel and detected with a rabbit monoclonal antibody (Cell Signaling).

Cell cycle progression and Fluorescence Activated Cell Sorting (FACS) analysis profiles of AAV2 infected breast cancer cell lines. Cells were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 106 cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase. This time point was designated time zero (t = 0). (A) MCF-7 (B) MDA-MB-468 and (C) MDA-MB-231 cells were infected with AAV2 at this point, and further cultured over a 7-day period. Both control and AAV2 infected cells were passaged 1:2 on day 2 and day 5. On each day, cells were harvested by trypsinization, washed with PBS, fixed in 70% ethanol, and stored at -20°C for 24 h. For performing FACS analysis, cells were resuspended in PBS containing 0.1% Triton X-100, 200 μg/ml DNase-free RNase A, and 100 μg/ml of propidium iodide for 30 min at 37°C. Flow cytometric analysis of 106 cells was carried out in a fluorescence-activated cell sorter, and the percentages of cells in theG1, S and G2/M phases of the cell cycle were determined using the Cell Quest program of Becton Dickinson. Data were analyzed with the Mod Fit LT program. For each cell line, FACS analyses were repeated three times. Results shown represent averages determined from three individual experiments (-, no data), with standard deviations presented in parentheses.

AAV2 infection of different breast cancer cells and induction of apoptosis/cell death results in altered expression profiles of different cell cycle tumor suppressors proteins. (A) MCF-7 (B) MDA-MB-468 and (C) MDA-MB-231 monolayer cell cultures were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 106 cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase followed by infection with AAV2. This time point was designated time zero (t = 0). Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of cell cycle tumor suppressor proteins was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. The pRb protein hyperphosphorylated (Hyper-pRb) and hypophosphorylated (hypo-pRb) forms were detected using the rabbit polyclonal antibody (Santa Cruz). The p21WAF1, p27KIP1 and p16INK4 tumor suppressors were detecting using rabbit polyclonal antibodies (Santa Cruz) as described in Materials and Methods. The p53 protein was detected using a mouse monoclonal antibody (Oncogene).

AAV2 infection of different breast cancer cell lines and induction of apoptosis/cell death results in altered expression of proliferation markers PCNA, Ki67 and c-Myc. (A) MCF-7 (B) MDA-MB-468 (C) MDA-MB-231 and (D) CIN-612 9E (HPV31b positive cervical cancer cells) monolayer cell cultures were synchronized by trypsinization of 80% confluent cultures and plating at a density of 1 × 106 cells in E medium. The cells were incubated for 10 to 12 h at which point at least two-thirds of the cells are maximally synchronized in the G1 phase followed by infection with AAV2. This time point was designated time zero (t = 0). Cell pellets were collected each day over a 7 day period. Cells were passaged 1:2 on day 2 and day 5. Detection of cell cycle proliferation proteins was performed by Western blotting. Total protein extracts were prepared as described previously [48]. Sixty micrograms of total protein extracts from AAV2 infected and control cells were resolved in SDS-polyacrylamide gel electrophoresis (PAGE) gels. The PCNA protein was detected using a rabbit polyclonal antibody (Santa Cruz). The Ki67 protein was detected using a rabbit polyclonal antibody (Santa Cruz). The c-Myc protein was detected using a rabbit polyclonal antibody (Santa Cruz).